Biological indicator for use with vaporous microbial deactivating agents and method for making same

ABSTRACT

A biological indicator and method of making same. The biological indicator includes a carrier having a recess formed therein in order to restrict movement of an inoculum deposited onto the carrier. The inoculum includes microorganisms (e.g., bacterial spores) suspended in a suspension medium. The microorganisms are prepared by removing extraneous material and subjecting the microorganisms to sonication to break up agglomerations. The suspension medium includes a wetting agent to reduce surface tension, thereby facilitating flow of the suspension medium to prevent stacking of microorganisms on the surface of the carrier, and to allow the inoculum to more evenly “plate out” on carrier surfaces. The carrier, with inoculum deposited thereon, is enclosed in an envelope made of a material permeable to a vaporous deactivating agent (e.g., vaporized hydrogen peroxide, ozone, chlorine dioxide, ethylene oxide, etc.), thereby facilitating exposure to the vaporous deactivating agent.

FIELD OF THE INVENTION

The present invention relates to a biological indicator for determiningthe efficacy of a microbial deactivation process.

BACKGROUND OF THE INVENTION

Many articles (e.g., medical instruments and devices) and enclosedregions of a wide range of facilities (e.g., medical treatment andresearch facilities, pharmaceutical manufacturing facilities, animalresearch facilities, laboratories, patient rooms, hotel rooms, offices,cruise ships, recreational facilities and vehicles) are treated with avaporous deactivating agent (e.g., vaporized hydrogen peroxide) in amicrobial deactivation process to deactivate microbial contamination onthe articles or contaminated surfaces within the enclosed region. Inorder to determine whether the treated articles or enclosed regions havebeen successfully deactivated, and thereby safe for use, it is necessaryto determine whether all of the parameters necessary for deactivationwere met during the deactivation process or are present within theenclosed region during the microbial deactivation process. To this end,biological indicators accompany the articles, or are located within theenclosed region, throughout the microbial deactivation process. Atypical biological indicator includes a known number of microorganisms(usually bacterial spores) of known resistance to the mode ofdeactivation, located in or on a carrier (also referred to as a “coupon”or “strip”), and enclosed in a protective package. Before themicroorganisms are deposited onto the carrier, the microorganisms aretypically suspended in a suspension medium. Subsequent growth or failureof the microorganisms to grow, i.e., after the deactivation process,under suitable conditions indicates the efficacy of the microbialdeactivation process.

Known biological indicators include a carrier formed of a metal, such asstainless steel. The carrier has a uniform flat surface on one side ofthe carrier, upon which microorganisms are deposited. The carrier istypically enclosed within the protective package having one side formedof a material permeable to a vaporous deactivating agent (e.g., Tyvek®)and having the other side formed of a material impermeable to thevaporous deactivating agent (e.g., Mylar®). The carrier is orientedwithin the package such that the side of the carrier having themicroorganisms thereon faces the permeable side of the package, whilethe opposite side of the carrier faces the impermeable side of thepackage.

One problem with known biological indicators is that microorganismsbecome “stacked” on the surface of the carrier, thereby shielding someof the microorganisms from exposure to the vaporous deactivating agent.FIG. 1 illustrates a prior art biological indicator 70 comprised of acarrier 72 having microorganisms 78 suspended within a suspension medium76. Suspension medium 76 is deposited onto the flat upper surface ofcarrier 72. As shown in FIG. 1, microorganisms 78 are “stacked” withinsuspension medium 76 due to suspension medium 76 failing to more evenlydistribute across the upper surface of carrier 72.

Still another problem encountered with known biological indicators isthat microorganisms disposed on the flat upper surface of the carriercome into contact with the package enclosing the carrier. As a result,microorganisms can be removed from the carrier. Once removed from thecarrier, microorganisms may migrate to the opposite side of the carrierfacing the impermeable packaging. As a result, the microorganisms may be“masked” from the deactivation process.

Yet another problem with known biological indicators is that the carriermay shift positions within the protective package, thereby causing theside of the carrier having microorganisms deposited thereon to face theimpermeable side of the protective package. Accordingly, exposure of themicroorganisms to the vaporous deactivating agent is inhibited.

The problems described above result in a biological indicator that doesnot accurately indicate the efficacy of a microbial deactivationprocess.

The present invention overcomes these and other problems by providing animproved biological indicator for determining the efficacy of amicrobial deactivation process using a vaporous deactivating agent, anda method for making said biological indicator.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a biological indicator for determining the efficacy of amicrobial deactivation process used to deactivate items by exposing saiditems to a vaporous deactivating agent, said biological indicatorcomprising: a carrier having a recess formed therein; and an inoculumincluding microorganisms suspended in a suspension medium, wherein theinoculum is deposited in the recess.

In accordance with another aspect of the present invention, there isprovided a method of making a biological indicator comprised of acarrier and an inoculum including microorganisms suspended in asuspension medium, the biological indicator determining the efficacy ofa deactivation process used to deactivate articles by exposing saidarticles to a vaporous deactivating agent, said method comprising thesteps of: (a) cleaning the carrier using a cleaning agent including adetergent; (b) removing extraneous material from said microorganisms;(c) exposing said microorganisms to ultrasonic vibrations to break upagglomerations of said microorganisms; (d) suspending saidmicroorganisms in the suspension medium to form the inoculum; and (e)depositing said inoculum onto said carrier.

An advantage of the present invention is the provision of a biologicalindicator having an inoculum with minimal extraneous material therein.

Another advantage of the present invention is the provision of abiological indicator having a suspension medium that minimizes stackingof microorganisms.

Still another advantage of the present invention is the provision of abiological indicator having a carrier dimensioned to inhibit migrationof inoculum.

Still another advantage of the present invention is the provision of abiological indicator having a carrier formed of a material compatiblewith an oxidative vaporous microbial deactivating agent.

Still another advantage of the present invention is the provision of abiological indicator having a protective packaging with improvedpermeability for vaporous microbial deactivating agents.

Yet another advantage of the present invention is the provision of amethod for making the above-mentioned biological indicator.

Yet another advantage of the present invention is the provision of amethod of making a biological indicator that minimizes agglomeration ofmicroorganisms.

These and other advantages will become apparent from the followingdescription of a preferred embodiment taken together with theaccompanying drawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may take physical form in certain parts and arrangement ofparts, a preferred embodiment of which will be described in detail inthe specification and illustrated in the accompanying drawings whichform a part hereof, and wherein:

FIG. 1 is a perspective cutaway view of a carrier of a prior artbiological indicator;

FIG. 2 is a perspective cutaway view of a carrier for a biologicalindicator according to an embodiment of the present invention;

FIG. 3 is a cross-sectional view of a biological indicator according toan embodiment of the present invention, said biological indicatorincluding a carrier, inoculum and packaging.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

It should be understood that as used herein the term “vaporous”deactivating agents also includes “gaseous” deactivating agents. By wayof example, and not limitation, the deactivating agents may includevaporized hydrogen peroxide, ozone, chlorine dioxide, and ethyleneoxide.

Referring now to the drawings wherein the showings are for the purposeof illustrating a preferred embodiment of the invention only, and notfor the purpose of limiting same, FIG. 3 shows a biological indicator(BI) 10 according to an embodiment of the present invention. BI 10 isgenerally comprised of a carrier 20 (best seen in FIG. 2); an inoculum30 comprised of a plurality of microorganisms 38 suspended in asuspension medium 36; and an envelope 46. Inoculum 30 is prepared bysuspending microorganisms 38 within suspension medium 36. Inoculum 30 isdeposited onto carrier 20. Thereafter, carrier 20 is sealed withinenvelope 46. Each component of BI 10 is described in detail below.

In the illustrated embodiment, carrier 20 is a generally planar plate orstrip having a cavity or recess 22 formed on one side thereof, as bestseen in FIG. 2. Recess 22 is defined by a generally planar bottomsurface 24 and a plurality of side walls 26 surrounding bottom surface24. Side walls 26 slope upward from bottom surface 24 to a generallyplanar upper surface 28 that is disposed around the periphery of recess22. Recess 22 is dimensioned to receive inoculum 30, comprised ofmicroorganisms 38 suspended in a suspension medium 36.

Carrier 20 is preferably formed of a polymeric material such aspolypropylene, polyethylene, polyvinyl-carbonate, polyvinyl styrene,polyvinyl chlorine, polyethylene terephthalate, nylon, epoxy, or amixture of polymeric materials. It is also contemplated that carrier 20may be formed of a metal (such as aluminum or stainless steel) or aceramic. However, carrier 20 is preferably formed of a polymericmaterial since polymeric materials do not oxidize in the presence ofoxidizing vaporous microbial deactivating agents, such as vaporizedhydrogen peroxide; provide flexibility to allow a wide variety of shapesfor carrier 20; and are relatively inexpensive.

In a preferred embodiment, suspension medium 36 of inoculum 30 is anaqueous solution comprising an oxidant-compatible surfactant. Suitablesurfactants include, but are not limited to, Triton® X-100, Tween® 80 orsodium dodecyl sulfate. The surfactant acts as a wetting agent to reducesurface tension, thereby reducing microorganism “stacking.” Suspensionmedium 36 may also include an alcohol to increase the rate at whichsuspension medium 36 dries after being deposited into recess 22 ofcarrier 20. Suitable alcohols include, but are not limited to, ethylalcohol or iso-propyl alcohol. Reduced surface tension and faster dryingallows suspension medium 36, and microorganisms 38 suspended therein, tomore easily and readily spread across bottom surface 24 of carrier 20.Increased spreading of inoculum 30 minimizes “stacking” ofmicroorganisms 3 8 within suspension medium 36.

In the illustrated embodiment, microorganisms 38 of inoculum 30 arebacterial spores, including, but not limited to, the following:Geobacillus stearothermophilus, Bacillus atrophaeus, B. pumilus, C.sporogenes, and combinations thereof. It should be understood thatmicroorganisms 38 are not limited to bacterial spores, and thus mayinclude bacteria (such as Staphylococcus aureus, Salmonella choleraesuisand Pseudomonas aeruginosa), fungi (such as Tricophyton mentagrophytes),and viruses.

In the illustrated embodiment, envelope 46 is comprised of two generallyflexible, planar sheets 48 a and 48 b. Sheets 48 a, 48 b are attached toeach other along their perimeters (e.g., by an adhesive or by heatsealing). Envelope 46, formed by sheets 48 a and 48 b, is dimensioned toenclose carrier 20. In the illustrated embodiment sheets 48 a, 48 b areboth formed of a material permeable to vaporous deactivating agents,such as vaporized hydrogen peroxide, but generally impermeable tomicroorganisms, such as bacterial spores. Preferably, sheets 48 a, 48 bare formed of a nonwoven polymer material, such as an olefin that isspun-bonded. A suitable spun-bonded olefin material is commerciallyavailable from Dow Chemical as Tyvek®. In one embodiment of the presentinvention, sheets 48 a, 48 b are formed of 2FS Tyvek®. By way of exampleand not limitation, sheets 48 a, 48 b may also be formed of one of thefollowing materials: 1073B Tyvek®, other Tyvek® products, non-wovenpolymers, olefins, polyester film, and a combination thereof. However,2FS Tyvek® is a preferred material due to its permeability with respectto vaporous deactivating agents.

Preparation of the surfaces of carrier 20 in advance of depositinginoculum 30 thereon will now be described. Carrier 20 is prepared asfollows: during a cleaning step, carrier 20 is exposed to a cleaningagent for a predetermined period of time. Preferably, the cleaning agentincludes an enzymatic detergent, such as STERIS Klenzyme® detergent,available from STERIS Corporation of Mentor, Ohio. The purpose of thecleaning step is to remove oils (e.g., casting oils), organics, dirt andother contaminants from the surface of carrier 20, thereby reducingoxidant demand during a microbial deactivating process. In this regard,contaminants, such as oil, can be oxidized by a vaporous decontaminatingagent (e.g., vaporized hydrogen peroxide), thus reducing the amount ofvaporous deactivating agent acting upon the target microorganisms 38.

Following the cleaning step, carrier 20 is exposed to a rinsing fluidfor removal of any residual materials remaining on the surfaces ofcarrier 20, such as residual cleaning agent. In one embodiment, therinsing fluid includes an alcohol such as ethyl or isopropyl alcohol.

Prior to suspension within suspension medium 36, microorganisms 38undergo a “washing” procedure to remove extraneous material therefrom.By way of example, but not limitation, the extraneous material mayinclude cellular debris, excess organic material, growth media, and“spent” microorganisms 38. Removal of extraneous material frommicroorganisms 38 also reduces oxidant demand during a microbialdeactivating process. As a result, a greater amount of the vaporousdeactivating agent can act upon the target microorganisms 38. It shouldbe understood that dyes are preferably omitted from suspension medium 36to reduce oxidative demand during a microbial deactivating process,since dyes are also inherently prone to oxidative attack.

As indicated above, removing contaminants from the surfaces of carrier20, removing extraneous materials from microorganisms 38 prior todeposition onto carrier 20, and eliminating dyes from suspension medium36, minimizes the oxidative demand from materials other thanmicroorganisms 38. Accordingly, a greater amount of the oxidativevaporous deactivating agent is available to act on microorganisms 38within suspension medium 36. As a result, the accuracy of BI 10 isimproved.

Prior to suspension within suspension medium 36, microorganisms 38 alsoundergo a “mild” sonication (e.g., ultrasonic vibrations) to minimizeagglomeration (i.e., “clumping”) of microorganism 38. Reduction ofagglomeration improves the ability of microorganisms 38 to flow orspread across bottom surface 24 of carrier 20 when inoculum 30 isdeposited in recess 22.

After suspension medium 38 and microorganisms 36 have been prepared asdescribed above, inoculum 30 is formed by suspending microorganisms 36in suspension medium 38.

Following preparation of inoculum 30 and carrier 20, as described above,inoculum 30 is deposited into recess 22 of carrier 20. Inoculum 30spreads out across bottom surface 24 due to the reduced surface tensionprovided by the surfactant. Side walls 26 maintain inoculum 30 withinrecess 22, thereby preventing migration of inoculum 30 onto uppersurface 28. Side walls 26 also prevent inoculum 30 from becoming removedfrom carrier 20 or from migrating onto the inner surfaces of envelope46. In addition, side walls 26 minimize “drop formation” by causinginoculum 30 to plate out on side walls 26. Recess 22 allows fasterproduction of BI 10, since migration of inoculum 30 is inhibited.

As indicated above, the reduced surface tension of suspension medium 36facilitates the spreading of inoculum 30 across the area of bottomsurface 24. As a result, microorganisms 38 are spread across a largersurface area than with prior art BI 70, thereby minimizing stacking ofmicroorganisms 38. Less stacking of microorganisms 38 results inimproved exposure of microorganisms 38 to the vaporous deactivatingagent. In this regard, when microorganisms 38 are stacked (see FIG. 1),microorganisms 38 located at the top of the stack block microorganisms38 located below. The blocked microorganisms 38 thus fail to be properlyexposed to the vaporous deactivating agent. As a result, the accuracy ofthe biological indicator can be impaired.

The presence of the alcohol in suspension medium 36 allows for fastdrying of inoculum 30 after deposition into recess 22, therebyminimizing opportunities for unwanted migration of inoculum 30 duringhandling of BI 10. The presence of alcohol also reduces surface tension,thereby providing the advantages described above.

After inoculum 30 has dried on bottom surface 24 of carrier 20, carrier20 is sealed inside envelope 46. As indicated above, sheets 48 a, 48 bare preferably formed of a material permeable to the vaporousdeactivating agent (e.g., vaporized hydrogen peroxide), but impermeableto microorganisms 38.

Operation of BI 10 will now be described with reference to thedeactivation of articles within a deactivation chamber. BI 10 is placedwithin the deactivation chamber along with articles that are to besubject to a microbial deactivation process. The articles and BI 10 areexposed to a vaporous deactivating agent (such as vaporized hydrogenperoxide) during the microbial deactivation process.

After the microbial deactivation process is completed, biologicalindicator 10 is removed from the deactivation chamber and microorganisms36 are cultured to determine whether any of microorganisms 36 remainviable following the microbial deactivation process. If less than athreshold number of microorganisms 36 remain viable following exposureto the vaporous deactivating agent during the microbial deactivationprocess, then the microbial deactivation process is considered to havebeen effective.

The foregoing is one embodiment of the present invention. It should beappreciated that this embodiment is described for purposes ofillustration only, and that those skilled in the art may practicenumerous alterations and modifications without departing from the spiritand scope of the invention. It is intended that all such modificationsand alterations be included insofar as they come within the scope of theinvention as claimed or the equivalents thereof.

1. A biological indicator for determining the efficacy of a microbialdeactivation process used to deactivate items by exposing said items toa vaporous deactivating agent, said biological indicator comprising: acarrier having a recess formed therein; and an inoculum includingmicroorganisms suspended in a suspension medium, wherein the inoculum isdeposited in the recess.
 2. A biological indicator as defined in claim1, wherein said recess is defined by a bottom surface and sloping sidewalls that surround the bottom surface and slope away therefrom.
 3. Abiological indicator as defined in claim 1, wherein said microorganismsinclude at least one of the following: bacterial spores, bacteria,fungi, viruses, or a combination thereof.
 4. A biological indicator asdefined in claim 1, wherein said biological indicator further comprisesan envelope for enclosing said carrier having said inoculum depositedthereon.
 5. A biological indicator as defined in claim 4, wherein saidenvelope is formed of a material that is permeable to said vaporousdeactivating agent and generally impermeable to said microorganisms. 6.A biological indicator as defined in claim 5, wherein said materialcomprises spun-bonded olefin.
 7. A biological indicator as defined inclaim 1, wherein said suspension medium includes a wetting agent.
 8. Abiological indicator as defined in claim 7, wherein said wetting agentis a surfactant compatible with said vaporous deactivating agent.
 9. Abiological indicator as defined in claim 1, wherein said suspensionmedium includes an alcohol.
 10. A biological indicator as defined inclaim 1, wherein said carrier is formed of a polymeric material.
 11. Abiological indicator as defined in claim 10, wherein said polymericmaterial includes at least one of the following: polypropylene,polyethylene, polyvinyl-carbonate, polyvinyl styrene, polyvinylchlorine, polyethylene terephtlialate, nylon, epoxy, or a mixture ofpolymeric materials.
 12. A biological indicator as defined in claim 1,wherein said carrier is formed of a metal.
 13. A biological indicator asdefined in claim 12, wherein said carrier includes at least one of:aluminum or stainless steel.
 14. A biological indicator as defined inclaim 1, wherein said carrier is formed of a ceramic.
 15. A biologicalindicator as defined in claim 1, wherein said vaporous deactivatingagent is vaporized hydrogen peroxide.
 16. A method of making abiological indicator comprised of a carrier and an inoculum includingmicroorganisms suspended in a suspension medium, the biologicalindicator determining the efficacy of a deactivation process used todeactivate articles by exposing said articles to a vaporous deactivatingagent, said method comprising the steps of: cleaning the carrier using acleaning agent including a detergent; removing extraneous material fromsaid microorganisms; exposing said microorganisms to ultrasonicvibrations to break up agglomerations of said microorganisms; suspendingsaid microorganisms in the suspension medium to form the inoculum; anddepositing said inoculum onto said carrier.
 17. A method of making abiological indicator as defined in claim 16, wherein said step ofremoving extraneous material from said microorganisms includes washingsaid microorganisms.
 18. A method of making a biological indicator asdefined in claim 16, wherein said suspension medium includes a wettingagent.
 19. A method of making a biological indicator as defined in claim18, wherein said wetting agent is a surfactant compatible with saidvaporous deactivating agent.
 20. A method of making a biologicalindicator as defined in claim 16, wherein said suspension mediumincludes an alcohol.
 21. A method of making a biological indicator asdefined in claim 16, wherein said method further comprises: enclosingsaid carrier having inoculum deposited thereon in an envelope made of amaterial permeable to said vaporous deactivating agent and generallyimpermeable to said microorganisms.
 22. A method of making a biologicalindicator as defined in claim 21, wherein said material includes aspun-bonded olefin.
 23. A method of making a biological indicator asdefined in claim 16, wherein said method further comprises the step of:rinsing said carrier with a rinsing solution including an alcohol, priorto depositing said inoculum thereon.
 24. A method of making a biologicalindicator as defined in claim 16, wherein said microorganisms include atleast one of the following: bacterial spores, bacteria, fungi, viruses,or a combination thereof.
 25. A method of making a biological indicatoras defined in claim 1, wherein said vaporous deactivating agentincludes: vaporized hydrogen peroxide, ozone, chlorine dioxide, orethylene oxide.